Effects of Diet on Plumage Coloration and Pigment Deposition in Red And

bioRxiv preprint doi: https://doi.org/10.1101/027532; this version posted September 24, 2015. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

RRH: Koch et al. CANARY CAROTENOID PIGMENTS

In review: Wilson Journal of Ornithology

EFFECTS OF DIET ON PLUMAGE COLORATION AND PIGMENT DEPOSITION IN RED

AND YELLOW DOMESTIC CANARIES

REBECCA E. KOCH1,3, KEVIN J. MCGRAW2, GEOFFREY E. HILL1

1 Department of Biological Sciences, 331 Funchess Hall, Auburn University, Auburn, AL 36849

2 Arizona State University, School of Life Sciences, Life Sciences C-wing Rm. 522, Tempe, AZ

85287-4501

3 Corresponding Author; e-mail: [email protected]

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ABSTRACT.--- The Atlantic Canary (Serinus canaria) is the most common caged bird with

extensive carotenoid plumage coloration. Domestic strains of canaries have been bred for a range

of colors and patterns, making them a valuable model for studies of the genetic bases for feather

pigmentation. However, no detailed account has been published on the feather pigments of the

various strains of this species, particularly in relation to dietary pigments available during molt.

Moreover, in the twentieth century, aviculturists created a red canary by crossing Atlantic

Canaries with Red Siskins (Carduelis cucullata). This “red-factor” canary is reputed to

metabolically transform yellow dietary pigments into red ketocarotenoids, but such metabolic

capacity has yet to be documented in controlled experiments. We fed molting yellow and red-

factor canaries seed diets supplemented with either β-carotene, lutein/zeaxanthin, or β-

cryptoxanthin/β-carotene and measured the coloration and carotenoid content of newly grown

feathers. On all diets, yellow canaries grew yellow feathers and red canaries grew red feathers.

Yellow canaries deposited dietary pigments and metabolically derived canary xanthophylls into

feathers. Red-factor canaries deposited the same plumage carotenoids as yellow canaries, but

also deposited red ketocarotenoids. Red-factor canaries deposited higher total amounts of

carotenoids than yellow canaries, but otherwise there was little effect of diet treatment on feather

hue or chroma. These observations indicate that canaries can use a variety of dietary precursors

to produce plumage coloration and that red canaries can metabolically convert yellow dietary

carotenoids into red ketocarotenoids.

Key words: domestic canary, carotenoid pigments, plumage coloration, carotenoid conversion bioRxiv preprint doi: https://doi.org/10.1101/027532; this version posted September 24, 2015. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

In most birds, red, orange, and yellow plumage coloration is produced by carotenoid

pigments (Goodwin 1984, McGraw 2006). No birds can synthesize carotenoids de novo, but

some birds can biochemically modify the carotenoids that they ingest (Brush 1981). The most

common carotenoids in the diets of most birds are lutein, zeaxanthin, β-carotene, and β-

cryptoxanthin, and some birds convert these dietary pigments into yellow canary xanthophylls or

red ketocarotenoids (carotenoids containing a ketone group) that they deposit into feathers or

bare parts (McGraw 2006). Although these metabolic transformations have been deduced for a

handful of birds (Brush 1990, McGraw 2006), such data are surprisingly lacking for the Atlantic

Canary (Serinus canaria), the most common caged bird with extensive carotenoid plumage

coloration.

In the wild on the Canary Islands, Atlantic Canaries have yellow feathers on their heads

and breasts, with mostly brown wing, tail, and dorsal plumage (Collar et al. 2010). This

appearance is retained in some domestic canary varieties, particularly those bred for singing

ability. In the so-called “color-bred canaries,” however, a fantastic range of color patterns and

novel shades of red, orange, and yellow have been generated through selective breeding (Walker

and Avon 1993, Birkhead 2003). In our study of feather pigments, we focused on the simple

lipochrome canaries, which have bright carotenoid-based coloration distributed more-or-less

uniformly across the plumage with little or no melanin pigmentation of feathers (Walker and

Avon 1993). Yellow lipochrome canaries have uniform bright yellow coloration across their

plumage. Red-factor lipochrome canaries, which originated in the early 1900s by crossing yellow

lipochrome canaries with Red Siskins (Carduelis cucullata; Birkhead 2003, 2014), have uniform

red coloration across their plumage. Although red-factor canaries are a hybrid taxon, the hybrid

offspring were back-crossed with canaries with each generation selected for red coloration but no bioRxiv preprint doi: https://doi.org/10.1101/027532; this version posted September 24, 2015. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

other siskin characteristics (Birkhead 2003). Thus, individuals of this breed retain no

measureable remnants of siskin phenotype except red plumage coloration.

Within the pet trade, canaries are often “color fed” by adding high doses of carotenoid

pigments to the diet of molting birds to induce an especially brilliant plumage color; in fact, even

yellow lipochrome canaries may acquire an orange tint upon consumption of a diet exceptionally

high in red ketocarotenoids, though only red-factors may acquire truly red coloration (Birkhead

2003, Birkhead 2014). However, the diets of seed-eating songbirds like canaries typically

contain exclusively yellow or orange pigments that must be converted into red pigments for

ornamental coloration (Hill 2006). Isolating the red-factor canary’s ability to metabolize yellow

dietary carotenoids into red carotenoids in the absence of color feeding is essential to

understanding the differences in carotenoid processing between red and yellow canaries, and

may have important implications generally for the control and evolution of red carotenoid

metabolism in birds and other animals.

Canaries are important birds for ornithological research. They are model animals for song

and neurobiological research (Goldman and Nottebohm 1983, Brenowitz and Beecher 2005), and

strains have been developed with genetic knockouts for carotenoid pigmentation (white canaries)

as well as for color variants (red, yellow, orange, frosted, dark red, etc.; Walker and Avon 1993).

Canaries could serve as a model for studies of the genetics of carotenoid plumage coloration

(Pointer et al. 2012, Walsh et al. 2012), and a first step in this process is deducing what feather

pigments are produced from what dietary precursors. In the only published experimental study of

feather pigmentation in domestic canaries, Brockmann and Völker (1934) detected canary

xanthophylls in the feathers of canaries held on a seed diet, but did not examine the metabolic

processes occurring within these birds. Modern carotenoid analytical techniques are now needed bioRxiv preprint doi: https://doi.org/10.1101/027532; this version posted September 24, 2015. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

to substantiate and build on the results of this study, which was one of the first to quantify

carotenoid content in the feathers of any bird.

Here, we assessed the effect of diet on plumage pigmentation in yellow and red-factor

lipochrome canaries by feeding molting red and yellow canaries different carotenoid-containing

diets during plumage molt and measuring the pigments deposited in newly grown feathers. Our

goal was to demonstrate, under controlled experimental conditions, that red-factor canaries can

convert yellow dietary carotenoids into red plumage pigments and to determine the range of

dietary precursors from which canaries can produce different metabolic plumage pigments.

METHODS

All canaries were housed as pairs in 0.46 by 0.46 by 0.92 m cages illuminated with full-

spectrum lights set to the natural photoperiod of Auburn, AL. Birds were fed a maintenance diet

of primarily canary seed (All Natural Canary Blend, Jones Seed Company), which provides

sufficient nutrition for canaries to molt and to breed, but has low levels of yellow dietary

carotenoids (<20 µg/g, lutein with low levels of zeaxanthin and β-carotene; Brockmann and

Völker 1934) that are well below the supplemented levels (see more below). Birds of both sexes

were included in the experiment because domestic canaries are sexually monochromatic (Koch

and Hill in press) .

In July 2014, we randomly divided 18 red-factor and 18 yellow color-bred lipochrome

canaries (Marvin Walton, The Canary’s Nest), evenly split between both sexes, equally into three

treatment groups that were supplemented with different yellow dietary carotenoids:

lutein/zeaxanthin (FloraGLO, The Vitamin Shoppe), β-carotene (β-Carotene 15, GNC), or

ground freeze-dried papaya (Freshlydried, Jersey City, NJ). Papaya was used as a source of β- bioRxiv preprint doi: https://doi.org/10.1101/027532; this version posted September 24, 2015. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

cryptoxanthin, though it also provided birds with some β-carotene; red papaya has about four

times the concentration of β-cryptoxanthin as β-carotene (Chandrika et al. 2003). We chose these

diets because they provided birds with all four common carotenoids in the diets of songbirds,

with each of the four dietary carotenoids supplied in abundance in at least one dietary treatment.

Three birds in the lutein/zeaxanthin treatment died before the start of the experiment, so our final

sample sizes were 4 red and 5 yellow in the lutein/zeaxanthin treatment, 6 red and 6 yellow in

the β-carotene treatment, and 6 red and 6 yellow in the papaya treatment.

Carotenoids for all treatments were applied as powder to seed by adding about 3 g

carotenoids to 20 g of canary seed. By this method of supplementation, we had no way to

determine exactly how much carotenoids per day were ingested by birds. However, our methods

were sufficient for the goals of this study: to determine whether red or yellow canaries have the

physiological capability to convert dietary yellow carotenoids to red ketocarotenoids, and to

make deductions about which dietary carotenoid are suitable precursors for carotenoid

conversion.

Birds received experimental supplementation throughout the duration of their annual fall

molt, ending in October 2014. We monitored birds for growing feathers, and removed 6-10

newly grown breast feathers for color measurement and carotenoid analysis. We stacked feather

samples on nonreflective black cardstock paper and measured their color using an Ocean Optics

USB4000 spectrophotometer and the OOIBase32 program according to standard color

measurement protocols (Saks et al. 2003). From the raw reflectance spectra, we used the

program CLR (v. 1.05; Montgomerie 2008) to calculate one value of hue (variable name H3) and

one value of chroma (also known as saturation; S3); we selected these variables because they

have been shown to most accurately reflect the carotenoid content of feathers in another bioRxiv preprint doi: https://doi.org/10.1101/027532; this version posted September 24, 2015. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

cardueline finch that can display both red and yellow plumage, the House Finch (Haemorhous

mexicanus; Butler et al. 2011).

We assessed the carotenoid content of each bird’s feather sample using high-performance

lipid chromatography (HPLC) according to the methods described in Friedman et al. (2014).

While our primary goal was to qualitatively describe the types of carotenoids present or absent

within canaries of each treatment and color type, we also used analysis of variance (coupled with

post-hoc tests using Tukey’s Honest Significant Difference method), t-tests, and linear regression

models in R (version 3.0.2; R Core Team 2015) to investigate possible quantitative relationships

between diet treatment, plumage color, and feather carotenoid concentrations within red and

yellow canaries.

RESULTS

Based on human visual categorization, all red-factor canaries produced only orange and

red feathers (never yellow) and all yellow lipochrome canaries produced only yellow feathers,

regardless of diet treatment. Yellow lipochrome canaries deposited only yellow canary

xanthophylls and lutein into feathers, whereas red-factor canaries deposited yellow canary

xanthophylls, lutein, and red ketocarotenoids into plumage (Table 1; Appendix). Thus, red but

not yellow canaries were able to produce red ketocarotenoids from both carotenes (carotenoids

containing only carbon and hydrogen; e.g. β-carotene) and hydroxycarotenoids (carotenoids

containing a hydroxyl group, e.g. zeaxanthin) precursors (Fig. 1).

Red canaries had higher total feather carotenoid concentration than yellow canaries

across all treatments (F1,27 = 6.84, P = 0.014; Table 1; Fig. 2). However, there was no significant

difference in concentration of yellow plumage carotenoid pigments between red and yellow bioRxiv preprint doi: https://doi.org/10.1101/027532; this version posted September 24, 2015. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

canaries (F1,31 = 0.93, P = 0.34; Table 1), so differences in total carotenoids appeared primarily

due to the extra presence of red carotenoids in their feathers.

There were no significant effects of diet treatment on plumage hue, saturation, total

carotenoid concentration, total yellow carotenoid concentration, or total red carotenoid

concentration (for red canaries only; all P > 0.16), with the exception that red-factor canaries on

the β-carotene treatment had significantly more total carotenoids than yellow canaries on the

same treatment (t = 3.30, df = 6.06, P = 0.016; Table 1; Fig. 2). However, we found that the total

carotenoid concentration of canary feathers significantly affected plumage saturation (t = 5.30, df

= 30, P < 0.001), but not hue (t = 1.56, df = 30, P = 0.13), in both color types. Saturation is

therefore a better predictor than hue of feather carotenoid content in this species.

DISCUSSION

Wild-type canaries deposit canary xanthophylls in their feathers to produce the species-

typical yellow feather coloration (Stradi 1999). In the early twentieth century, aviculturists

crossed Atlantic Canaries with Red Siskins to produce canaries capable of growing red feathers

(Birkhead 2003, Birkhead 2014), and all domestic canaries with red plumage are descended from

such siskin-canary crosses (Walker and Avon 1993). Although how red-factor canaries were

genetically engineered is well known (Birkhead 2003, Birkhead 2014), the dietary bases and

plumage pigment pathways of red or yellow domestic canaries have not be described (Stradi

1998, McGraw 2006). Thus, the studies presented here represent the first controlled experimental

tests of the hypothesis that red lipochrome canaries produce red ketocarotenoids from yellow

dietary pigments. bioRxiv preprint doi: https://doi.org/10.1101/027532; this version posted September 24, 2015. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

On any of the carotenoid diets that we provided, which varied from containing

xanthophylls (e.g. lutein/zeaxanthin) to carotenes (e.g. β-carotene), yellow canaries deposited

canary xanthophylls A, B, and C as well as lutein in their feathers. Thus, regardless of substrate

(those four most common in the diets of terrestrial birds and ranging from yellow to orange),

these birds could not metabolize dietary pigments into red plumage pigments. In addition, we

found that red-factor canaries can convert a diverse array of dietary precursors—namely β-

carotene, lutein/zeaxanthin, and β-cryptoxanthin—into the red ketocarotenoids α-doradexanthin,

canthaxanthin, and echinenone. These data are the first experimental demonstration that red

canaries can produce red ketocarotenoids from yellow dietary precursors alone.

Because we did not carefully control the quantities of carotenoids consumed by birds in

our experiments, comparisons of feather carotenoid concentrations across diet treatments must be

considered cautiously. Nevertheless, the data present some interesting patterns. When fed the

same diets, red canaries had significantly more total carotenoids in their feathers than yellow

canaries. In contrast, red and yellow canaries did not differ in yellow plumage carotenoid

concentration, so the difference in total feather carotenoid levels was due to the addition of red

pigments to the yellow pigments present in both canary strains. This observation implies that the

pathway for ketolation of red pigments in canaries does not work from the end point of the

pathway for canary xanthophylls, in which case yellow pigments would be depleted and would

exist in red birds in lower concentrations. Rather, it seems that the red ketolation pathway runs in

parallel to the yellow hydroxylation pathway (Hill and Johnson 2012). Parallel conversion

pathways likely explain our finding that in canary plumage carotenoid concentration predicts

saturation but not hue of feathers. Hue has been found to reflect the ratio of red to yellow

carotenoid pigments in feathers of House Finches , which do not use parallel conversion bioRxiv preprint doi: https://doi.org/10.1101/027532; this version posted September 24, 2015. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

pathways (Inouye et al. 2001); the lack of a relationship between carotenoid pigment

concentration and hue in the canary indicates that the feathers contain a generally uniform ratio

of red and yellow pigments in feathers, indicative of parallel conversion pathways. Saturation, on

the other hand, tends to reflect the absolute concentrations of carotenoid pigments in feathers

(Hill and McGraw 2006).

None of the treatments resulted in significant differences in total feather carotenoid

concentrations of either yellow or red birds, except for the β-carotene treatment on which red

birds had more total carotenoids than yellow bird. It is likely the lack of precision in carotenoid

delivery veiled differences in how various carotenoid precursors were used to produce feather

pigments, but our results do show that a range of pigment precursors can enable birds to reach

the same display endpoint. This study is the first to establish that domestic red-factor, but not

yellow, canaries can metabolize yellow-orange dietary pigments into red ketolated pigments.

ACKNOWLEDGMENTS

We would like to thank R. Montgomerie for development of the CLR program. Several

Auburn University undergraduates and graduate students (A. Halasz, X.R. Franko) helped with

canary husbandry and supplementation. R.E. Koch was supported by NSF GRFP throughout the

project.

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TABLE 1. Average carotenoid content (±SD) of feathers from red or yellow canaries on

one of three supplemental carotenoid treatments: β-carotene, lutein and zeaxanthin, or β-

cryptoxanthin and β-carotene. All carotenoid measurements are in units of ng carotenoids per g

feather; hue is in units of nm, such that higher values indicate redder color; saturation is a

unitless ratio calculated from the feather reflectance spectra such that higher values indicate

more saturated color. “Yellow carotenoids” comprises lutein, canary xanthophylls A, B, and C,

and xanthophyll isomers generated from these pigments during extraction; “Red carotenoids”

comprises echinenone, canthaxanthin, α-doradexanthin, and ketocarotenoid isomers generated

during extraction.

Color Yellow Red Total Treatment Hue Saturation type carotenoids carotenoids carotenoids

222.8 ± 367.6 ± 590.4 ± 441.8 ± 0.374 ± Red β-Carotene 82.5 244.0 296.4 120.0 0.047

170.0 ± 310.8 ± 480.8 ± 408.0 ± 0.368 ± Lutein/zeax. 61.7 391.2 446.1 103.6 0.021

β-Crypt./ 185.3 ± 121.3 ± 306.6 ± 422.0 ± 0.361 ±

β-Carotene 99.0 67.8 164.0 97.9 0.021

170.1 ± 170.1 ± 330.6 ± 0.337 ± Yellow β-Carotene 0 97.1 97.1 29.4 0.021

229.2 ± 229.2 ± 364.2 ± 0.324 ± Lutein/zeax. 0 207.4 207.4 25.1 0.021

β-Crypto./ 320.3 ± 0 320.3 ± 386.5 ± 0.340 ± bioRxiv preprint doi: https://doi.org/10.1101/027532; this version posted September 24, 2015. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

β-Carotene 161.4 161.4 63.7 0.026

FIGURE CAPTIONS

FIG. 1. Diagram illustrating the chemical structures and relevant conversion pathways of

four dietary carotenoids (lutein, zeaxanthin, β-cryptoxanthin, and β-carotene) into the

metabolically altered carotenoids detected in canary feathers (canary xanthophylls A, B, and C,

echinenone, canthaxanthin, and α-doradexanthin). The letters above the arrows indicate the

nature of the reaction undergone: D = dehydrogenation, H = hydroxylation, and O = oxidation

(McGraw 2006).

FIG 2. The average total feather carotenoid concentration (± SD) in the feathers of yellow

and red canaries fed one of three supplemental carotenoid treatments: β-carotene, lutein and

zeaxanthin, or β-cryptoxanthin and β-carotene.

FIG. 1.

FIG. 2.

APPENDIX. The concentrations in ng of all carotenoids assessed by HPLC and the hue

and chroma spectrographic values of the feathers of yellow or red-factor lipochrome canaries fed

one of three supplemental dietary treatments: β-carotene, lutein and zeaxanthin, or β-

cryptoxanthin and β-carotene.